The present study is the first to show that treatment (i.m.) with flaxseed oil for two weeks increases the contractile response induced through phenylephrine in the rat aorta. This response is endothelium-dependent, and most likely reflects an increase of COX-2-derived TXA2 and superoxide anion production.
Flaxseed oil has recently attracted much research attention, as this seed is one of the richest vegetable oil sources of ALA, associated with cardiovascular benefits, and has lower amounts of LA; these compounds are derived from the omega-3 (C18:3 n-3) and omega-6 (C18:2 n-6) families, respectively [4–6, 16].
Flaxseed oil contains n-3 ALA, a precursor molecule for the endogenous synthesis of f eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) . LA, which is present in lower amounts in flaxseed oil, is a precursor molecule for the synthesis of arachidonic acid (AA) . Some reports have demonstrated that EPA and DHA prevent the development of hypertension [18, 19] and exhibit anti-atherothrombogenic effects . These effects might be induced, at least in part, through a decrease in TXA2 and an increase in PGI3 in vessels, such as the aorta [10–12]. Therefore, we investigated the effects of flaxseed oil treatment on endothelial modulation in the vascular responses induced through α-adrenoceptor activation in isolated aortic rings, as the aorta is a major artery susceptible to atherosclerosis.
In the present study, we observed increased vascular reactivity to phenylephrine in isolated aortic rings from flaxseed oil-treated rats. However, the treatment did not impair the endothelium-dependent relaxation induced through acetylcholine. Similarly, the vascular response to sodium nitroprusside was unaffected, suggesting that the endothelial-independent routes through which vascular relaxation is modulated were unaltered.
The effects of flaxseed on contractile and vasodilator responses are controversial. A previous report showed that feeding with a flaxseed-supplemented diet for 6, 8 or 16 weeks does not modify norepinephrine-induced vasoconstriction or the relaxation response induced through acetylcholine or sodium nitroprusside . However, other reports have shown that a high-flaxseed diet enhances endothelial vasorelaxant function without reducing blood pressure in hypertensive rats . Flaxseed is a rich source of plant lignans. The lignin secoisolariciresinol diglucoside in flaxseed possesses potent antioxidant effects . Therefore, the improved endothelial vasodilatation, as previously demonstrated , could reflect the activities of lignans. However, Ogawa et al.  demonstrated that spontaneously hypertensive rats (SHR) fed a diet containing 10% flaxseed oil for four weeks did not show altered endothelium-dependent aortic relaxation in response to acetylcholine, although a significantly lower systolic blood pressure was detected.
To investigate the role of the endothelium in the increased phenylephrine responses induced through flaxseed oil treatment in aortic rings, the experiments in the present study were performed in the absence of endothelium. Endothelium removal promoted an increase in the phenylephrine-induced contraction in both groups, but this effect was smaller in the aortic rings from treated rats, suggesting that flaxseed oil affects endothelial function. These results suggest that the ability of the endothelium to negatively modulate the contractile response induced by phenylephrine may be impaired in isolated aortic rings of flaxseed oil-treated rats.
The increased vascular reactivity to phenylephrine and concomitant reduction of endothelial modulation in the aortic rings of treated rats might reflect reduced NO bioavailability . Therefore, L-NAME was used to investigate the putative role of NO in the effects of flaxseed oil treatment on the contractile responses induced through phenylephrine. The results (Figure 3) showed that the phenylephrine responses were similar in the aortic rings of both control and treated rats, suggesting that the flaxseed oil treatment did not affect the endothelial-induced NO modulation of the vasoconstrictor response. The data concerning the effects of flaxseed oil on the nitric oxide pathway is limited and controversial. Sekine et al.  demonstrated that the oral administration of 1 mL flaxseed oil for 5 days reduces systolic blood pressure and increases prostaglandin I2 (PGI2) and NO release in SHR. However, Karaca and Eraslan  showed that 0.1 ml flaxseed oil administered through gavage for 30 days did not alter nitric oxide levels in the heart, brain and liver of rats.
Flaxseed oil is rich in ALA. ALA and LA are synthesized through a series of elongation and desaturation reactions and can subsequently be incorporated into cell membrane phospholipids . EPA, DHA and AA act as alternative substrates for both cyclooxygenase (COX) and lipoxygenase (LOX) enzyme complexes, generating series 3 prostaglandins and thromboxanes and series 5 leukotrienes (EPA + DHA) or series 2 prostaglandins and thromboxanes and series 4 leukotrienes (AA) . The series 2 prostaglandins and thromboxanes increase vascular tone. Therefore, to assess the participation of these compounds, we used indomethacin to assess whether vasoconstrictor prostanoids are involved in the increased vascular reactivity to phenylephrine induced through flaxseed oil treatment. Indomethacin reduced the response to phenylephrine in aortic segments from both groups, but this reduction was greater in the aortic rings of flaxseed oil-treated rats, suggesting the increased participation of the COX pathway. Moreover, NS 398, SQ 29.548 and furegrelate reduced the vasoconstrictor responses to phenylephrine in aortic segments from treated rats, suggesting that vasoconstrictor prostanoids, specifically TXA2, increase reactivity to phenylephrine induced through flaxseed oil treatment. Consistent with these results, we also observed that COX-2 protein expression was greater in flaxseed oil-treated rats than in control rats.
Tou et al.  demonstrated that female rats fed a diet supplemented with flaxseed oil for 8 weeks showed decreased liver AA, and no significant differences in the series 2 eicosanoids, PGE2 and TXB2 metabolites were observed. However, Lee et al.  showed that the aortic production of PGI2 and the serum concentration of TXA2 were significantly low in rats fed a diet containing flaxseed oil for 4 weeks. However, Rupp et al.  showed an increased production of 6-keto-PGFlα, a PGI2 by-product, in rats fed a diet supplemented with flaxseed oil, although the arachidonic acid content was greatly reduced. Sekine et al.  reported similar results, showing that the plasma 6-keto-PGFlα levels were significantly higher in the fed flaxseed oil diet group.
PGI2 is synthesized in response to the release of the n-6 fatty acid AA from membrane phospholipids . However, in addition to the increased PGI2, the n-6 LA could also increase other series 2 prostanoids, such as TXA2, a potent vasoconstrictor . Moreover, a previous report demonstrated that high levels of PGI2 could induce a vasoconstrictor response through the activation of the TP receptor . Although we did not measure levels of PGI2 in the present study, this hypothesis cannot be discarded.
Moreover, eicosanoids derived from PUFA, such as AA and EPA, are physiologically active compounds that act locally as signaling molecules through G protein-linked receptors. When AA is predominantly incorporated into cell membrane phospholipids under conditions, such as injury or inflammation, phospholipase A2 might release AA. Subsequently, intracellular signaling cascades might elicit a wide range of responses, including vasoconstriction, the activation of leukocytes, the stimulation of platelet aggregation and the generation of reactive oxygen species . These effects might increase vulnerability to endothelial dysfunction and induce inflammation in the vessel wall, which is a key factor in atherosclerosis .
Thus, we suggest that the increased TXA2 induced through flaxseed oil treatment (i.m.) might increase phenylephrine contractions, as observed in the present study, through n-6 LA. Alterations in the eicosanoid profile might have important effects on inflammation . The n-3 and n-6 PUFA compete for COX and LOX enzymes. Moreover, the series 2 and series 4 eicosanoids derived from n-6 PUFA are more biologically active than the series 3 and series 5 eicosanoids derived from n-3 PUFA , even when present in smaller amounts . Furthermore, series 2 and series 4 eicosanoids can induce pro-inflammatory and pro-aggregatory states , likely promoting increased vascular reactivity .
Therefore, we used the phospholipase A2 blocker, dexamethasone, to investigate whether phospholipase A2 mediates the effects induced through flaxseed oil treatment in vascular reactivity. Dexamethasone abolished the effects of flaxseed oil on the contractile response to the α1-agonist, suggesting that a 15-day flaxseed oil treatment could increase phospholipase A2 activity, which in turn, increases eicosanoid production, leading to increased series 2 and series 4 eicosanoid activity. The effect of dexamethasone could be mediated through a reduction of COX-2-derived TXA2 release in response to α-adrenergic activation, as previously reported .
Glucocorticoids play an important role in the control of vascular smooth muscle tone through the modification of vasoconstrictor responses to different vasoactive agents and through alterations in vascular prostanoid and/or nitric oxide production [36, 37]. Therefore, dexamethasone could inhibit COX-2 in the isolated aortic rings of flaxseed oil-treated rats, normalizing vascular reactivity to the control group level. In addition to COX-2-derived products, the increased oxidative stress could also promote vascular hyperreactivity and endothelial dysfunction in aortic rings isolated from treated rats. The results demonstrated that apocynin, a ROS scavenger, and tiron, a cell permeant non-enzymatic scavenger of O2.–, decreased vascular reactivity to phenylephrine in aortic rings isolated from treated rats, suggesting that flaxseed oil exposure increases ROS, primarily O2.–, and induces oxidative stress.
Consistent with these findings, we observed an increase of local superoxide anion production in the aortic rings from flaxseed oil-treated rats. However, incubation with catalase did not alter the vascular contractile responses in both groups, suggesting that flaxseed oil treatment did not affect the release of hydrogen peroxide. Martínez-Revelles et al.  demonstrated that COX-2-derived products modulate ROS production and also, ROS can activate COX expression. Thus, we suggest that COX-2 prostanoids might increase ROS production and ROS might act on COX-2 expression, although these COX-2-derived products and ROS could operate independently.
However, to investigate whether the flaxseed oil treatment could induce an inflammatory state, we evaluated serum C-reactive protein levels. Our results demonstrated that the flaxseed treatment did not modify this parameter. Flaxseed oil has variable effects on the inflammatory mediators that seem to be dose- and time- dependent. Lower doses do not affect TNF-a, IL-1b, IL-6, or soluble intracellular adhesion molecule-1 (sICAM-1) in healthy adults . However, higher concentrations of flaxseed oil decrease the levels of cytokines . Moreover, 4 months of flaxseed oil supplementation appears to reduce the C-reactive protein levels .
A previous report demonstrated that flaxseed oil treatment increases lipid peroxidation and oxidative stress and reduces SOD activity . Another report showed that flaxseed oil does not affect catalase activity or superoxide dismutase in monkey livers in vivo and in vitro. Although we did not measure SOD activity, these results suggest that flaxseed oil treatment increased ROS production, primarily the superoxide anion, without increasing hydrogen peroxide.
However, although the present study demonstrated increased ROS production, a recent study showed that the combined effect of flaxseed oil and astaxanthin, a natural antioxidant, in rats fed a high-fat diet efficiently ameliorates oxidative stress, lipid profile and inflammation .
However, as mentioned above, flaxseed oil was also reported to promote and increase lipid peroxidation . Consistently, Nestel et al.  demonstrated that ALA reduces HDL and increases LDL plasmatic levels, likely reflecting high intake of ALA and increased vulnerability of polyunsaturated fatty acids to oxidation. This effect might alter the negative modulation of vascular tone in the endothelium, as shown in the present study.
In conclusion, the present study demonstrated that flaxseed oil treatment increases the vascular reactivity to phenylephrine and is associated with increased ROS production and the increased participation of COX-2 derivatives. Moreover, this treatment could increase phospholipase A2 participation, which in turn could increase the production of COX-2 inflammatory derivatives. Thus, the results of the present study provide new insights into the effects of flaxseed oil on vascular function.